The invention relates generally to the field of delivery of drugs, vectors, and other molecules in a variety of settings, including therapeutic, diagnostic, research and clinical uses.
Small organic molecules (i.e., those having molecular weights <10,000, and frequently <5,000 or <1,000) have well known uses as drugs in a variety of human ailments. Peptide and oligonucleotide drugs (including, for example, antisense oligonucleotides, ribozymes, aptamers, and siRNA/shRNA molecules) have emerged as powerful tools for drug target validation and promising therapeutics for a wide variety of human diseases. However, realization of the promise these agents offer as drugs has been hindered by the lack of efficient methods of delivering them to physiological sites at which they may exert useful activity. Obstacles to efficient delivery include considerations of in vivo stability, tissue and cell specificity, intracellular durability, immunogenicity, and toxicity of the agents. Many well-characterized small chemical drugs confront the same delivery bottlenecks that limit their potency in human applications.
Similarly, inorganic drugs (e.g., radioisotopes and radioisotope-containing compounds), imaging agents, and other small molecules intended for delivery to cells exhibit many of the same drawbacks with regard to their stability and delivery to desired cells and tissues.
Human Serum Albumin
Human serum albumin (HSA) is the most abundant protein in human plasma. HSA is known to bind an extraordinarily wide range of metabolites and drugs. Binding between HSA and such compounds affects, sometimes dramatically, their pharmacokinetics and pharmacodynamics.
HSA is synthesized in the liver and secreted as a non-glycosylated protein. It accounts for 60% of the mass of the plasma proteins and is present in the blood at a concentration around 0.6 mM with an average half-life of 14 days. Although the critical function of HSA in maintaining normal colloid osmotic pressure in plasma and in interstitial fluid is recognized, the molecular mechanisms of its basic physiological functions, such as metabolite transportation, exogenous chemical binding, and antioxidant protection, are not fully understood.
HSA is synthesized as a 585-residue single chain globular protein lacking prosthetic groups and glycosylation. The primary amino acid sequence of HSA is shown in FIG. 1. HSA has three homologous domains (designated domains I, II and III, as indicated in FIG. 2) that fold into the shape of a heart and each domain is classified into two subdomains (A and B). Further details of the structure of domains I, II, and III are shown in FIG. 3. Alpha-helixes account for approximately 67% of the secondary structure of HSA, and no beta-sheet secondary structure occurs in HSA. A unique feature of HSA is its 35 cysteine residues, 34 of which form 17 disulfide bonds. The only free cysteine (Cys-34, i.e., the cysteine residue that occurs at residue 34 of HSA) contributes significantly to the antioxidant activity of HSA and this residue can be chemical modified in a variety of known ways. The disulfide bonds are also responsible for the thermal stability of HSA. Others have recognized that the primary polypeptide structure of HSA includes several loops that are tightly held by disulfide bonds and present on the external surface of the protein.
Almost every body fluid contains some amount of HSA. In addition, HSA occurs within cells like ovarian cells, brain cells, peripheral nerve cells, lymphocytes, macrophages, and other cells. Tumor cells often take up HSA to a greater extent than non-tumorous cells of the same type. For example, albumin makes up 19% of the soluble protein of breast cancer cells.
Due to its availability, biocompatibility, nontoxicity, and immunogenicity, human serum albumin (HAS) has been used as a stabilizer in biopharmaceutical products (vaccine and recombinant protein), an adjuvant in drug formulation and a component of imaging agents. It also can be used to coat biomaterial surface and purify chiral chemicals. Moreover, adding signal peptides or functional compounds to albumin by chemical modification has been commonly used for drug targeting and delivery research. Using chemical crosslinking, HSA can also be formulated into microspheres and nanoparticles to encapsulate drugs, oligonucleotides, and radioisotopes for delivery or diagnosis purpose. However problems associated with the chemical modification may severely hinder the clinical application of chemically modified albumins. First, the modification is non-specific and non-homogenous. Second, these modifications change the physicochemical and biochemical properties of albumins and may result in an immune response. Third, the chemically modified albumin may not fold properly and exhibit an abnormal surface charge distribution. These changes are likely to be recognized by endogenous albumin and other serum proteins and cause aggregation and rapid elimination in vivo.
Long Chain Fatty Acids
Long chain fatty acids (LCFAs, i.e., carboxylic acids having an non-branched aliphatic chain having 16-20 carbon atoms in its backbone) are essential for many cellular functions. LCFAs serve as an important energy resource and are also critical components of lipids, hormones, and proteins. LCFAs are known to be bound and transported by HSAs within the human body.
Formation of conjugates between LCFAs and many small molecules is known to enhance the serum stability and delivery of the small molecules by a mechanism facilitated by binding of the small molecule-LCFA conjugate with HSA.
Shortcomings in stability, solubility, and ‘targetability’ limit the utility of many potentially useful drugs, diagnostic agents, nucleic acid vectors, and other relatively small molecules within the human body. The technology disclosed herein overcomes these shortcomings.